Sirt4 Lipoamidase Activity and Uses Thereof

Description:

A Novel Assay for Sirtuin 4 (SIRT4) Lipoamidase Activity and the Activity of the Pyruvate Dehydrogenase Complex

Princeton Docket # 14-3002

A novel assay for measuring the lipoamidase activity of SIRT4 in mammalian cells and tissue samples has been created.  A procedure by which the lipoamide cofactor activity can be monitored in mammalian cells has been developed.  Candidate agents to modulate the lipoamide cofactor can be identified with this research tool.

 

Sirtuins (SIRTs) are a family of seven mammalian nicotinamide adenine dinucleotide (NAD)-dependent enzymes that govern genome regulation, metabolism, and aging.  Despite conserved deacetylase domains, SIRTs 4-7 have little to no deacetylase activity, and robust catalytic activity for mitochondrial SIRT4 has remained elusive.  Princeton researchers in the Department of Molecular Biology have established SIRT4 as a cellular lipoamidase.  SIRT4’s catalytic efficiency for lipoyl- and biotinyl-lysine modifications is superior to its deacetylation activity.  Among its important substrates is the pyruvate dehydrogenase complex (PDH), which is regulated by SIRT4 through hydrolysis of its lipoamide cofactors.  SIRT4 lipoamidase activity is demonstrated to inhibit PDH activity, highlighting SIRT4 as a critical regulator of cellular metabolism.

 

Applications:   

·         Detect SIRT4 lipoamidase activity and relative abundance in cells and tissues

·         Discover inhibitors, activators, and substrates of SIRT4 lipoamidase activity

·         Identify candidate modulators of pyruvate dehydrogenase (PDH) complex

·         Identify small molecules and antibodies that regulate lipoyl modification on lysine residues

·         Implications for cancer, aging, mitochondrial diseases, viral infection, and cell signaling

·         Determine cellular pathways regulated by SIRT4

Advantages: 

·         Provides several assays for monitoring SIRT4 activity and for discovering substrates, including mass spectrometry, western blotting, fluorometric, and steady state kinetic assays

·         Proteomics approach avoids difficulties in maintaining soluble, active recombinant SIRT4 for in vitro assays

·         Accurately identifies regulators of PDH activity in any cell or tissue using targeted mass spectrometry with selected reaction monitoring

·         Accurately and quantitatively identify precise residues that are modified

·         High-throughput PDH activity assay

Publications

Mathias, R.A., Greco, T.M., Oberstein, A., Budayeva, H.G., Chakrabarti, R., Kang, Y., Shenk, T. and Cristea, I.M.  Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.  Cell.  2014, 159(7): 1615-1625.

 

 

Key Words

Sirtuins, SIRT4, lipoamidase activity, pyruvate dehydrogenase complex, metabolism, regulator

 

Inventors

Ileana M. Cristea, Thomas E. Shenk, Todd M. Greco, Rommel A. Mathias, Adam Oberstein

Faculty inventors

 

Ileana M. Cristea, Associate Professor of Molecular Biology

Professor Cristea’s research lies at the interface of proteomics and virology, employing a multidisciplinary approach to integrate proteomics techniques with genomics, microscopy, and bioinformatics.  Current work in her laboratory is being undertaken using mass spectrometry to discover critical biological findings, including the identification of viral mechanisms of host manipulation and defense mechanisms hosts deploy to protect themselves from viral attack.

 

Dr. Ileana Cristea conducted her graduate research at the Michael Barber Center for Mass Spectrometry, University of Manchester, U.K., under the supervision of Simon Gaskell, and at the Toxicology Research & Development Department at GlaxoSmithKline, U.K.  She pursued postdoctoral work in the mass spectrometry laboratory of Brian Chait at The Rockefeller University.  Currently, Dr. Cristea is a member of the Executive Board of the US Human Proteome Organization, US-HUPO Education Committee, and the American Society for Mass Spectrometry Education Committee, and on the Editorial Boards of several journals.  She is the head instructor of the summer Proteomics Course at Cold Spring Harbor Laboratory.  She is the recipient of many accolades, including the NIDA Avant-Garde Director Pioneer Award for HIV/AIDS Research (2008), Human Frontiers Science Program Young Investigator Award (2009), Early Career Award in Mass Spectrometry from the American Chemical Society NJ Section (2011), the American Society for Mass Spectrometry Research Award (2012), and the Molecular & Cellular Proteomics Lectureship (2013).

 

Thomas E. Shenk, James A. Elkins Professor in the Life Sciences

Thomas Shenk, Ph.D., is the James A. Elkins Professor of Life Sciences in the Department of Molecular Biology at Princeton University. He is a virologist who has investigated gene functions and pathogenesis of adenovirus, a DNA tumor virus, and, more recently, human cytomegalovirus, a member of the herpes family of viruses.  Cytomegalovirus is the leading known infectious cause of birth defects, it is responsible for significant morbidity in people who become immunosuppressed, and there is suggestive evidence that it contributes to certain cancers and immune senescence.  His laboratory’s current areas of focus include the use of genetic and proteomic approaches for the dissection of cytomegalovirus gene functions and the cellular response to infection, as well as the development and analysis of models for study of viral latency.  Professor Shenk is a fellow of the American Academy of Microbiology and the American Academy of Arts and Sciences; and he is a member of the U.S. National Academy of Sciences and the U.S. Institute of Medicine.  He is a past president of the American Society for Virology and the American Society for Microbiology, and he served on the board of directors of Merck & Company for 11 years.  He currently serves as a member of the boards of directors of the Fox Chase Cancer Center, The Hepatitis B Foundation and Origen Therapeutics.

 

Intellectual Property Status

Patent applications are pending.  Princeton is seeking industrial collaborators for further development and commercialization of this technology.

 

Contact

Laurie Tzodikov
Princeton University Office of Technology Licensing • (609) 258-7256•
tzodikov@princeton.edu

 

Sarah Johnson

Princeton University Office of Technology Licensing • sajohnso@princeton.edu

 

 

Patent Information:
For Information, Contact:
Laurie Tzodikov
Licensing Associates
Princeton University
tzodikov@princeton.edu
Inventors:
Thomas Shenk
Todd Greco
Ileana Cristea
Rommel Mathias
Adam Oberstein
Keywords:
biologics
Biotechnology/Pharmaceuticals
genomics/proteomics (software)
virology